Fluorescence techniques are finding increasing application in chemical and biochemical and medical analyses. Fluorescence measurement methods are intrinsically extremely sensitive. They can offer at least the sensitivity of radiochemical methods without the hazards associated with radiation.
U.S. Pat. Nos. 4,150,295 and 4,058,732, issued on Apr. 17, 1979 and Nov. 15, 1977, respectively, and a chapter appearing at pages 67-80 of Immunofluorescence and Related Staining Techniques, Knapp, et al eds. (1978, Elsevier/North Holland Biomedical Press) disclose the general concept of fluorescently quantitating nonfluorescent species using fluorophores with long decay lifetimes in comparison with ambient fluorescence. In this method, an atomic scale fluorescent tag (fluorophore) is chemically covalently affixed to the individual molecules of a species. This species may be an organic target molecule itself or it may be a molecule essentially identical with a target molecule or it may be an antibody specific to a target molecule. After suitable procedures, dependent on the form of the assay, the tagged species are excited and, using time-gated techniques taught in the three references, their fluorescence measured. Using the magnitude of the observed fluorescence and a previously prepared fluorescence/. concentration standard curve, the amount of target is determined.
Fluorophores and tagged species useful in such a determination ideally have a long fluorescent decay lifetime and retain their ability to fluoresce throughout the period of the analysis. For sensitive assays it is also important that the fluorophore and its linkage to other species be stable even at very low concentrations such as in the range of nanograms/cc and lower such as even to femtograms/cc. For many immunoassays it is desirable that the fluorophore be water soluble so as to be reacted with antibodies in an environment which preserves immune reactivity.
The metal chelates of the present invention include within their number a family of species-bound ligands which possess all of these desired properties. In addition, they are simple and inexpensive to prepare.